For the past twelve years this laboratory has been interested in mechanisms of RNA synthesis in bacterial and bacteriophage systems. As a model, the expression of the genome of bacteriophage T3 within the host (E. coli), as well as in vitro has been studied. Subsequent to infection of the bacterium by the phage, a new DNA-dependent RNA polymerase which directs transcription of the "late" genes of the phage genome is induced. The phage polymerase, a single subunit protein of MW 105,000, shows absolute specificity for T3 DNA. With this template in vitro, it directs the synthesis (from one strand) of eight discrete RNA species, most of which have in vivo counterparts. These major transcripts all have the 5'-sequence, pppGpGpGpApPuPPuP and the 3'-sequence, pGp(Up)5G OH, indicating highly specific "Start" and "Stop" signals for the enzyme. Given the availability of modern techniques for restriction enzyme analysis, purification of defined DNA fragments, nucleic acid sequencing, we will further characterize initiation and termination of transcription by T3 RNA polymerase in the following ways: (a) Promoters and terminators for the phage polymerase will be precisely mapped; (b) Detailed DNA and RNA sequences of these promoters and terminators will be obtained; (c) Mechanisms of termination will be investigated, specifically with regard to the role of nucleic acid secondary structure at the termination sites. In addition to the above studies, we are also carrying out studies on the mechanism of action initiation factors in the formation of mammalian 40S and 80S initiation complexes using AUG and globin mRNA as templates. The control mechanisms operating at each step of initiation of protein synthesis will be studied.